The present invention relates to peptide synthesis and, in particular, to solid phase peptide synthesis, or a combination of solid and liquid phase peptide synthesis. Many methods for peptide synthesis are described in the literature (for examples, see U.S. Pat. No. 6,015,881; Mergler et al. (1988) Tetrahedron Letters 29: 4005-4008; Mergler et al. (1988) Tetrahedron Letters 29: 4009-4012; Kamber et al. (eds), Peptides, Chemistry and Biology, ESCOM, Leiden (1992) 525-526; Riniker et al. (1993) Tetrahedron Letters 49: 11065-11133; and Andersson et al. (2000) Biopolymers 55: 227-250).
In solid phase peptide synthesis (SPPS), an amino acid or peptide group is bound to a solid support resin. Successive amino acids are attached to the support-bound peptide until the peptide of interest is formed. After the desired peptide is formed, it is cleaved from the resin. This requires cleaving the attachment between the peptide and resin and thereafter recovering the cleaved peptide using a suitable recovery technique.
Amino acids from which peptides are synthesized tend to have reactive side groups as well as reactive terminal ends. When synthesizing a peptide, it is important that the amine group on one peptide react with the carboxyl group on another peptide. Undesired reactions of side groups or at the wrong terminal end of a reactant produce unwanted by-products. To minimize side reactions, it is common practice to block reactive side groups and terminal ends of reactants to help make sure that the desired reaction occurs.
For example, a typical solid phase synthesis scheme involves attaching a first amino acid to the support resin via the carboxyl moiety of the first amino acid (although some synthesis schemes attach the first amino acid via the amine group). This allows the amine group of the resin bound amino acid to couple with an additional amino acid. Therefore, the carboxyl moiety of a new amino acid reacts with the free amine group of the resin bound material. To avoid side reactions involving the amine group of the new amino acid, the amine group is blocked with a protecting group during the coupling reaction. Two well-known amine protecting groups are the tert-butyloxycarbonyl (BOC) group and the 9-fluorenylmethyl carbamate (FMOC) group. Many others also have been described in the literature. After coupling, the protecting group (usually BOC or FMOC) on the N-terminus of the resin bound peptide can be removed, allowing additional amino acids to be added to the growing chain in a similar fashion. Reactive side chain groups of the amino acid reactants and the resin bound peptide can also be blocked with side chain protecting groups and remain blocked throughout the synthesis.
After synthesis, some or all of the side chain protecting groups can be removed from the peptide product. When substantially all of the protecting groups are removed, this is referred to as global deprotection. Global deprotection can occur contemporaneously with cleaving or can be carried out later if the peptide is to be further processed, modified, coupled to additional peptides or other material, etc. Some cleaving reagents not only cleave the peptide from the support resin, but also cause global deprotection to occur at the same time. For example, the strongly acidic cleaving reagents associated with BOC chemistry tend to cause global deprotection at the time of cleaving. Using the FMOC strategy, however, allows cleavage of the peptide from the resin while allowing the side chain protecting groups to remain so that further reactions, such as fragment condensation can, occur without substantial interference from side chain groups. Thus, the peptide is cleaved in a protected state.
Typically, the yield of a peptide synthesized by solid phase peptide synthesis decreases with increasing length of the peptide chain, i.e., the longer the peptide chain, the more likely undesirable side products will be produced along with the desired peptide. Therefore, for particularly long peptides, the final peptide product is produced in fragments, which are then combined later to form the desired peptide product. For example, a hypothetical 75 amino acid peptide may be synthesized in three peptide fragments, each fragment synthesized separately by solid-phase peptide synthesis. The fragments consisting of amino acids 1-25, amino acids 26-50, and amino acids 51-75 can be synthesized separately, then combined in fragment condensation steps to form the complete 75 amino acid final peptide product.
The prior art methods of cleaving a peptide from the resin support in a protected state typically create byproducts having carboxylic acids. Carboxylic acids will interfere with the subsequent fragment condensation reaction, creating unwanted byproducts. The prior art methods solved this problem by including an additional step following cleavage to remove the unwanted carboxylic acids, usually by evaporation of the cleavage solution. This extra step and the solvents needed cost additional time, expense, and create waste which must be disposed of, creating further expense. Therefore, there is a need for a method of cleaving a peptide from a resin more easily, cheaply, and efficiently by avoiding the production of unwanted carboxylic acid byproducts and, thereby, avoiding subsequent steps that remove the carboxylic acids.